Impact of nicotinamide mononucleotide on transplanted mouse ovarian tissue.

Ovarian tissue cryopreservation and future transplantation is the only strategy to preserve the fertility of young female adolescent and prepubertal patients. The primary challenge to ovarian graft longevity is the substantial loss of primordial follicles during the period of ischaemia post-transplantation. Nicotinamide mononucleotide (NMN), a precursor of the essential metabolite NAD+, is known to reduce ischaemic damage. Therefore, the objective of the current study was to assess the impact of short- and long-term NMN administration on follicle number and health following ovarian tissue transplantation. Hemi-ovaries from C57Bl6 mice (n = 8-12/group) were transplanted under the kidney capsule of bilaterally ovariectomised severe combined immunodeficient (SCID) mice. Recipient mice were administered either normal drinking water or water supplemented with NMN (2 g/L) for either 14 or 56 days. At the end of each treatment period, ovarian transplants were collected. There was no effect of NMN on the resumption of oestrous or length of oestrous cycles. Transplantation significantly reduced the total number of follicles with the greatest impact observed at the primordial follicle stage. We report that NMN did not prevent this loss. While NMN did not significantly impact the proportion of apoptotic follicles, NMN normalised PCNA expression at the primordial and intermediate stages but not at later stages. In conclusion, NMN administration did not prevent ovarian follicle loss under the conditions of this study.

Closed vitrification of human oocytes and blastocysts: outcomes from a series of clinical cases.

PURPOSE: High survival rates and clinical outcomes similar to those from fresh oocytes and blastocysts have been observed with open oocyte vitrification systems. It has been suggested that the extremely fast cooling rates that are only achieved with open systems are necessary for human oocyte and blastocyst vitrification. However, there is a potential risk of introducing contamination with open systems. The aim of this study was to assess whether similar survival and subsequent implantation rates could be achieved using a closed vitrification system for human oocytes and blastocysts. METHODS: Initially, donated immature oocytes that were matured in vitro were vitrified using the cryoprotectants ethylene glycol (EG) + dimethyl sulphoxide (DMSO) + sucrose and either a closed system (Rapid-i®) or an open system (Cryolock). The closed system was subsequently introduced clinically for mature oocyte cryopreservation cases and blastocyst vitrification. RESULTS: Using in vitro matured oocytes, a similar survival was achieved with the open system of 92.4 % (73/79) and with the closed system of 89.7 % (35/39). For clinical oocyte closed vitrification, high survival rate of 90.5 % (374/413) and an implantation rate of 32.7 % (18/55) from the transfer of day 2 embryos was achieved, which is similar to fresh day 2 embryo transfers. Blastocysts have also been successfully cryopreserved using the Rapid-i closed vitrification system with 94 % of blastocysts having an estimated ≥75 % of cells intact and a similar implantation rate (31.5 %) to fresh single blastocyst transfers. CONCLUSION: Closed vitrification can achieve high survival and similar implantation rates to fresh for both oocytes and blastocysts.

Impact of oxygen concentration on adult murine pre-antral follicle development in vitro and the corresponding metabolic profile.

Oxygen concentration during in vitro culture has a significant effect on the physiology of embryos, altering metabolic profile and developmental outcome. Although atmospheric oxygen has been used routinely for the culture of ovarian follicles, oxygen concentration may also be critical for follicle growth but the optimal concentration has not been determined. In this study, mechanically isolated primary and secondary follicles (80-140 µm diameter) from adult mouse ovaries were cultured in serum-free conditions for 8 days in either 5 or 20% oxygen to determine growth (follicular diameter), morphology and viability. For each oxygen concentration, half of the medium was replaced on Days 2, 4 and 6 or on Day 4 only. In the latter group, metabolic analysis of spent follicular culture media was performed by (1)H-NMR. The proportion of viable, growing follicles was significantly (P < 0.0001) higher in 5% than in 20% oxygen (59% versus 8%). Reducing the frequency of medium replacement during culture in 5% oxygen resulted in significantly (P < 0.001) more viable follicles (79 versus 46%). In 20% oxygen, poor follicular viability was observed irrespective of the frequency of medium replacement (8 and 10% respectively). Metabolic profiles showed marked differences in amino acid and carbohydrate utilization with respect to both oxygen concentration and between Days 4 and 8 of development. Metabolites which significantly discriminated between oxygen concentration at both time points were glucose consumption, lactate utilization, alanine, alanyl-glutamine, leucine and proline. In conclusion, the poor in vitro follicular development previously observed in minimal culture conditions may reflect the use of 20% oxygen. Frequent medium replenishment is not necessary and does not overcome the detrimental effect of high oxygen on follicle viability. Further optimization of culture conditions would benefit from metabolic analyses and the use of 5% oxygen should be tested further for impact on functional aspects of follicle culture such as steroid production which is currently unknown.

History of oocyte cryopreservation.

The potential advantages of being able to cryopreserve oocytes have been apparent for many decades. Technical difficulties associated with the unique properties of the mammalian oocyte initially retarded rapid development in this area but recent advances have overcome many of the problems. A stage has now been reached where oocyte cryopreservation can be considered an important component of human assisted reproductive technology. The potential advantages of being able to cryopreserve oocytes have been apparent for many decades. Technical difficulties associated with the unique properties of the mammalian oocyte initially retarded rapid development in this area but recent advances have overcome many of the problems. A stage has now been reached where oocyte cryopreservation can be considered an important component of human assisted reproductive technology.

Implantation rates of embryos generated from slow cooled human oocytes from young women are comparable to those of fresh and frozen embryos from the same age group.

Previous reports of slow cooling of human mature oocytes have shown a reduced clinical efficiency relative to fresh oocytes. This study reports that equivalent fertilization and implantation rates to those obtained using fresh oocytes and cryopreserved embryos can be achieved with human mature oocytes dehydrated in 1.5 M propanediol and 0.2 M sucrose at 37°C and cryopreserved using slow cooling rates.

The first Australian experience of heterotopic grafting of cryopreserved ovarian tissue: evidence of establishment of normal ovarian function.

Cryostorage of reproductive potential, in the form of ovarian cortex, for young women about to undergo cytotoxic therapies has been offered clinically for some time. However, the prospects of re-establishing reproductive function using this tissue remain unclear. We now report reproducible follicular development, oocyte retrieval and embryo development following heterotopic grafting of cryopreserved ovarian cortex which had been stored for over 10 years.

Increasing dehydration of human cleavage-stage embryos prior to slow cooling significantly increases cryosurvival.

Increasing the proportions of embryos and blastomeres which survive cryopreservation would be expected to make a significant contribution to the outcome of assisted reproduction treatment. Despite this, the methodology used for slow cooling of human cleavage-stage embryos has remained largely unchanged for over two decades. Previous studies have demonstrated the value, in terms of cryosurvival, of increasing the extent of intracellular dehydration by increasing the concentration of non-permeating cryoprotectant prior to slow cooling of oocytes and embryos which have been biopsied for preimplantation genetic diagnosis. The present study extends the use of this approach to the slow cooling of non-biopsied day-2 embryos. Dehydration in the presence of 0.2 mol/l sucrose significantly increased the proportions of surviving embryos, surviving blastomeres and fully intact embryos (92.6%, 91.1%, and 80.4%, respectively) relative to those observed after dehydration in 0.1 mol/l sucrose (78.5%, 74.1%, and 54.6%, respectively, all P < 0.001). Post-thaw resumption of mitosis in vitro and implantation were not adversely affected by the increased prefreeze dehydration. This improved method for slow cooling of cleavage-stage embryos should have a major impact on clinical outcome.

Cryopreservation of female reproductive potential.

Storing female reproductive potential can offer enhanced prospects for future conception in women whose fertility is threatened by cytotoxic therapies. Human female reproductive potential can be cryopreserved and stored at very low temperatures as embryos or gametes. Gamete (oocyte) cryopreservation circumvents potential issues associated with ownership when future use is being considered and may, therefore, be more generally acceptable as an approach. Advances in the technology, in particular the clinical application of vitrification, have significantly improved the outcomes from mature oocyte cryopreservation, which are now comparable to those from embryo cryopreservation. In cases where mature oocyte cryopreservation is not feasible, ovarian cortex containing primordial follicles can be cryopreserved, and over 100 births have now been reported following grafting of stored ovarian tissue. Ovarian tissue cryopreservation is now an established approach to preserve future fertility for young women; however, the efficiency is difficult to determine particularly for the prepubertal tissue with a scarcity of data.

Characterization of follicles in girls and young women with Turner syndrome who underwent ovarian tissue cryopreservation.

OBJECTIVE: To characterize ovarian follicles of girls and young women with Turner syndrome (TS) who underwent ovarian tissue cryopreservation (OTC). DESIGN: Retrospective case-control study. SETTING: University hospital. PATIENT(S): Fifteen girls and young women with TS aged 5-22 years at OTC were included, together with 42 control girls and young women aged 1-25 years who underwent OTC because of cancer. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Follicle density (follicles/mm3), morphology, and health were assessed in ovarian cortex biopsies from TS patients and compared with controls. Hormone concentrations were measured in serum and follicle fluids. Immature cumulus oocyte complexes were obtained and matured in vitro. RESULT(S): Follicles were found in 60% of the biopsies (9 of 15) from TS ovaries. In 78% of the ovaries (7 of 9) with follicles, the follicle density was within the 95% confidence interval of the control group. There was a high rate of abnormal follicle morphology. Six follicle-specific proteins were expressed similarly in TS and control ovaries. However, apoptosis and zona pellucida protein expression were found to be abnormal in TS. Turner syndrome follicle fluid from small antral follicles had lower concentrations of estrogen and testosterone and higher concentrations of antimüllerian hormone than controls. Thirty-one cumulus oocyte complexes were collected from one patient and cultured for 48 hours in vitro, resulting in five metaphase II oocytes (maturation rate 16%, degeneration rate 19%). CONCLUSION(S): The benefits of OTC may be limited to a highly selected group of TS mosaic patients in whom a sizeable pool of normal follicles is present at OTC.

Detection of zona pellucida proteins during human folliculogenesis.

BACKGROUND: The stage of folliculogenesis at which the human zona pellucida (ZP) is initiated and the cells responsible for the origin of the ZP continue to be controversial. This study characterizes the development of the ZP during human folliculogenesis using ovarian samples donated from patients requesting ovarian storage. METHODS: Follicles (from n = 18 patients, 14-40 years old) within fresh tissue and following development in a xenograft system were stained, using immunohistochemical techniques, for the presence of the three human ZP proteins, ZP1, ZP2 and ZP3. Over 500 primordial follicles and >20 follicles at each developmental stage were examined. RESULTS: All three ZP proteins were detected within the oocyte of the primordial follicle. Presence of ZP1 and ZP3 was observed in the majority of primordial oocytes (93% and 95%, respectively), whereas ZP2 was detected in only 32% of these follicles. The three ZP proteins were detected in the cytoplasm of cuboidal granulosa cells and their distribution correlates with developmental stages throughout folliculogenesis. CONCLUSIONS: ZP proteins were detected in both the oocyte and the granulosa cells as early as the primordial follicle stage in the human. The detection of ZP proteins in the quiescent primordial follicle suggests that these proteins have been present since oogenesis.

Chapter 12 Human Ovarian Tissue Slow Freezing.

Cryopreservation of human ovarian tissue is now being accepted as a mainstream ART laboratory procedure. The procedure described has been validated for use at the histological and functional level, and, recently, unequivocal evidence of preservation of primordial follicles has been demonstrated following twin births from cryopreserved ovarian tissue grafted at a heterotopic site.

Chapter 9 Slow Freezing and Thawing of Human Cleavage Stage Embryos.

The ability to store human embryos in a viable state at very low temperatures has been critical to the evolution of responsible practice in clinical Assisted Reproductive Technology (ART). It has encouraged a reduction in the frequency of simultaneous multiple embryo transfer and thereby reduced the risks associated with multiple pregnancy while maintaining high cumulative pregnancy rates from single oocyte collection cycles. In this chapter, we describe a simple slow freezing procedure for human early cleavage stage embryos that results in a high proportion of post-thaw embryos surviving and retaining their implantation potential.

Recombinant human zona pellucida proteins ZP1, ZP2 and ZP3 co-expressed in a human cell line.

AIM: To produce biologically active recombinant human (rh) ZP proteins in a human cell for use in sperm function tests. METHODS: The human embryonic kidney cell line 293T was employed to produce rhZP1, rhZP2 and rhZP3 proteins individually and together by co-expression. Presence of these proteins in the culture medium and cell lysate was assessed by Western blotting analysis. The effect of the recombinant proteins on the human AR was assessed. RESULTS: RhZP2 and rhZP3 were secreted into the culture medium, whereas rhZP1 was found only in the cell lysate. Interestingly, when all zona pellucida proteins were co-expressed in the same cells, rhZP1 was also secreted into the culture medium. However, despite the presence of all three ZP proteins in sufficient concentration and evidence of heavy glycosylation on gel electrophoresis, biological activity to induce the AR was not observed. CONCLUSION: RhZP1, rhZP2 and rhZP3 were successfully expressed in the human embryonic kidney cell line 293T. It appears that an interaction amongst these proteins may be required for release of rhZP1 from the cell. Although this approach is not satisfactory for producing active human ZP proteins, it makes a significant contribution to the understanding of the structural and functional characteristics of the ZP proteins.

Cryopreservation of human ovarian tissue.

Approaches to the cryopreservation of human ovarian tissue are typically characterised by the use of slow freezing/rapid thawing methods using dimethyl sulfoxide or 1,2-propanediol (PROH) as cryoprotectants. This paper reviews current experience with these procedures.

Follicle development following cryopreservation of human ovarian tissue.

Despite the recent increase in human ovarian tissue banking, there has been little progress in establishing whether follicles within this tissue are viable and capable of function following cryopreservation. Two methods to assess growth and developmental potential of cryopreserved tissue are evaluated; (1). isolated follicle culture and (2). xenografting of tissue into a host animal. Development of numerous antral follicles following xenografting of cryopreserved tissue indicates that the cryopreservation procedure can preserve the developmental competence of primordial follicles.

A critical appraisal of cryopreservation (slow cooling versus vitrification) of human oocytes and embryos.

BACKGROUND: Vitrification is now a commonly applied technique for cryopreservation in assisted reproductive technology (ART) replacing, in many cases, conventional slow cooling methodology. This review examines evidence relevant to comparison of the two approaches applied to human oocytes and embryos at different developmental stages. METHODS: Critical review of the published literature using PubMed with particular emphasis on studies which include data on survival and implantation rates, data from fresh control groups and evaluation of the two approaches in a single setting. RESULTS: Slow cooling is associated with lower survival rates and compromised development relative to vitrification when applied to metaphase II (MII) oocytes, although the vitrification results have predominantly been obtained using direct contact with liquid nitrogen and there is some evidence that optimal protocols for slow cooling of MII oocytes are yet to be established. There are no prospective randomized controlled trials (RCTs) which support the use of either technique with pronuclear oocytes although vitrification has become the method of choice. Optimal slow cooling, using modifications of traditional methodology, and vitrification can result in high survival rates of early embryos, which implant at the same rate as equivalent fresh counterparts. Many studies report high survival and implantation rates following vitrification of blastocysts. Although slow cooling of blastocysts has been reported to be inferior in some studies, others comparing the two approaches in the same clinical setting have demonstrated comparable results. The variation in the extent of embryo selection applied in studies can lead to apparent differences in clinical efficiency, which may not be significant if expressed on a 'per oocyte used' basis. CONCLUSIONS: Available evidence suggests that vitrification is the current method of choice when cryopreserving MII oocytes. Early cleavage stage embryos can be cryopreserved with equal success using slow cooling and vitrification. Successful blastocyst cryopreservation may be more consistently achieved with vitrification but optimal slow cooling can produce similar results. There are key limitations associated with the available evidence base, including a paucity of RCTs, limited reporting of live birth outcomes and limited reporting of detail which would allow assessment of the impact of differences in female age. While vitrification has a clear role in ART, we support continued research to establish optimal slow cooling methods which may assist in alleviating concerns over safety issues, such as storage, transport and the use of very high cryoprotectant concentrations.

Detection of Hodgkin lymphoma within ovarian tissue.

OBJECTIVE: To describe the detection of Hodgkin lymphoma within ovarian tissue taken at the time of harvest for cryopreservation. DESIGN: Case report. SETTING: University-affiliated women's hospital. PATIENT(S): A 19-year-old woman diagnosed with Hodgkin lymphoma. INTERVENTION(S): Laparoscopic removal of ovarian tissue for cryopreservation. MAIN OUTCOME MEASURE(S): Histologic and immunohistochemical evaluation of ovarian tissue harvested for fertility preservation. RESULT(S): Histologic and immunohistochemical identification of Hodgkin lymphoma within ovarian tissue harvested for cryopreservation. CONCLUSION(S): Ovarian cryopreservation and subsequent autografting is a procedure still in an experimental phase that has yielded promising findings. This option is frequently offered to young women with neoplasms such as Hodgkin lymphoma. Although the risk of Hodgkin lymphoma infiltration into the ovary may be low, the identification of lymphoma in this case emphasizes the importance of histologic examination of ovarian tissue before freezing and indicates that there is a possibility of reintroducing tumor.

How should the clinical efficiency of oocyte cryopreservation be measured?

Clinical application of oocyte cryopreservation may be in the context of fertility preservation for women about to undergo cytotoxic therapies or may be as an alternative to embryo cryopreservation in routine assisted reproduction. The clinical efficiency of oocyte cryopreservation will be a consequence of the cumulative impact of pre-freeze oocyte quality, postthaw survival and subcellular effects of cryopreservation protocols, which impact on early embryo quality and post-transfer viability, together with the degree of selection which is applied to the available biological material. Any valid assessment must include reference to all the above aspects, particularly when comparing cryopreserved oocytes with non-frozen controls or cryopreserved embryos. Cumulative pregnancy rates from oocyte collections may provide the most relevant index of success. Survival of human oocytes cryopreserved using current methodology is similar to that achieved with early-cleavage-stage embryos. Although evidence suggests that developmental potential may be compromised when current oocyte cryopreservation protocols are applied, there is a paucity of rigorously controlled studies in the literature.

Human oocyte cryopreservation.

The clinical role of oocyte cryopreservation in assisted reproduction, as an adjunct to sperm and embryo cryopreservation, has been comparatively slow to evolve as a consequence of theoretical concerns related to efficacy and safety. Basic biological studies in the 1990's alleviated many of these concerns leading to more widespread adoption of the technology. While a number of babies were born from the approach validated in the 1990's, its perceived clinical inefficiency led to the search for improved methods. Introduction of elevated dehydrating sucrose concentrations during cryopreservation increased survival and fertilization rates, but there is no well-controlled evidence of improved clinical outcome. Similarly, the use of sodium-depleted cryopreservation media has not been demonstrated to increase clinical efficiency. More recently, and in the absence of basic biological studies addressing safety issues, the application of vitrification techniques to human oocytes has resulted in reports of a number of live births. The small number of babies born from clinical oocyte cryopreservation and the paucity of well-controlled studies currently preclude valid comparisons between approaches. Legal restrictions on the ability to select embryos from cryopreserved oocytes in Italy, where many of the available reports originate, also obscure attempts to assess oocyte cryopreservation objectively.